Circulating alpha-galactosidase A derived from transduced bone marrow cells: relevance for corrective gene transfer for Fabry disease.
Circulating alpha-galactosidase A derived from transduced bone marrow cells: relevance for corrective gene transfer for Fabry disease.
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源自转导骨髓细胞的循环 α-半乳糖苷酶 A：与法布里病校正基因转移的相关性。
作者：
T. Takenaka;G. Qin;R. Brady;J. Medin
法布里病是由溶酶体α -半乳糖苷酶a （α -半乳糖苷酶a）缺乏引起的。我们先前设计了一种逆转录病毒，编码人类α -半乳糖a，并证明了酶对患者细胞的纠正。此外，我们证明了代谢协同性，在校正后的细胞分泌α -半乳糖A，可以被体外的旁观者细胞吸收和利用。在本研究中，我们创建了一个系统来检查和量化这种现象在体内。为了与内源性α -半乳糖A区分，我们构建了一个逆转录病毒载体（pUMFG/ α -半乳糖A/FLAG），其中含有α -半乳糖A的融合形式，在羧基端具有特定的标签序列。融合蛋白的催化活性与野生型α -gal a相同，融合蛋白在转导的患者细胞中过表达并分泌。在摄取研究中，在受体细胞的溶酶体富集部分中检测到融合蛋白。然后，我们在体内检测了pUMFG/ α -g A/FLAG逆转录病毒载体的有效性。将小鼠骨髓（BM）细胞转导并移植到受辐照的宿主体内。9周后，用PCR检测外周血和骨髓单核细胞的前病毒DNA。更重要的是，可以在这些细胞和血浆中证明特定的融合蛋白酶活性。因此，我们已经证明，过度表达的α -半乳糖A从转导的骨髓细胞进入循环，并在相当长的一段时间内保持稳定。
Fabry disease is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). We previously engineered a retrovirus encoding human alpha-gal A and demonstrated enzymatic correction of patient cells. Further, we demonstrated metabolic cooperativity, in that corrected cells secrete alpha-gal A that can be taken up and utilized by bystander cells in vitro. In the present study, we created a system to examine and quantitate this phenomenon in vivo. To differentiate from endogenous alpha-gal A, we constructed a retroviral vector (pUMFG/alpha-gal A/FLAG) containing a fusion form of alpha-gal A with a specific tag sequence at the carboxy terminus. The catalytic activity of the fusion protein was identical to wild-type alpha-gal A. The fusion protein was overexpressed in and secreted by transduced patient cells. In uptake studies, the fusion protein was detected in the lysosome-enriched fraction of recipient cells. We then examined the effectiveness of the pUMFG/alpha-g A/FLAG retroviral vector in vivo. Murine bone marrow (BM) cells were transduced and transplanted into irradiated hosts. After 9 weeks, proviral DNA was detected by PCR in peripheral blood and BM mononuclear cells. More importantly, specific fusion protein enzymatic activity could be demonstrated in those cells and in plasma. Thus, we have demonstrated that overexpressed alpha-gal A enters the circulation from transduced BM cells and is stable over a significant period of time.
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对接受转导骨髓细胞的初次和二次移植法布里小鼠的多个器官进行长期酶校正和脂质减少。
DOI：
10.1073/pnas.120177997
发表时间：
2000
影响因子：
11.1
作者：
Takenaka,T;Murray,GJ;Qin,G;Quirk,JM;Ohshima,T;Qasba,P;Clark,K;Kulkarni,AB;Brady,RO;Medin,JA
通讯作者：
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DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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DOI：
--
发表时间：
1998
期刊：
BioTechniques.
影响因子：
--
作者：
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通讯作者：
Bost,KL